Monomeric Collagen Research Articles

Особенности формирования 3d-структур для регенеративной медицины на основе коллагена, пектина и акриловых мономеров в присутствии комплекса триэтилбора с гексаметилендиамином

Cross-linked grafted copolymers containing collagen, pectin and a synthetic polymer: polymethylmethacrylate or polyacrylamide in the presence of a complex of triethylboron with hexamethylenediamine in acetic acid dispersion at 60 °C have been synthesized. The proportion of the synthetic fragment in the copolymer and its molecular-weight characteristics depend on the monomer structure, the concentration of the initiator and the monomer. The results are important for the development of methods to produce 3D-structures for regenerative medicine based on collagen, pectin and acrylic monomers.

Simultaneous co-assembly of collagen and glycosaminoglycans to build a biomimetic extracellular matrix for bone regeneration

Glycosaminoglycans (GAGs), also known as shape modules, are considered junctions that help define the shape of collagen matrix and further promote mineralization during osteogenesis. Many attempts have been made to immobilize GAGs on assembled collagen to modify the latter's surface state. However, it remains unclear how GAGs spontaneously identify collagen molecules during fibrillogenesis in vivo. Understanding the relationship between GAGs and collagen from both the bone physiology and materials science perspectives is of fundamental interest. Here, we introduced hyaluronic acid (HA, a main member of GAGs) during collagen self-assembly, in a process called modification cooperating with self-assembly (MCS). The molecular docking and morphological studies revealed that HA can help define collagen monomer deposition and thus promote fibrillogenesis through steric hindrance or by directly forming hydrogen bonds. Meanwhile, HA acts as a templating chaperone (TC) to increase the local mineral concentration within intrafibrillar channels but does not initiate nucleation, thus improving the crystallinity of formed apatite. The scaffolds synthesized through MCS model significantly improved the physicochemical stability and mechanical strength of collagen-based scaffolds. The optimized scaffolds promoted in-situ osteogenesis by stimulating the osteogenic differentiation of bone mesenchymal stem cells, either in an osteogenic medium, or after implantation into critical calvarial defects. This study provides novel insights towards evolving engineering scaffolds from inert supports to functional substitutes.

Collagen-targeted protein nanomicelles for the imaging of non-alcoholic steatohepatitis

In vivo molecular imaging tools hold immense potential to drive transformative breakthroughs by enabling researchers to visualize cellular and molecular interactions in real-time and/or at high resolution. These advancements will facilitate a deeper understanding of fundamental biological processes and their dysregulation in disease states. Here, we develop and characterize a self-assembling protein nanomicelle called collagen type I binding – thermoresponsive assembled protein (Col1-TRAP) that binds tightly to type I collagen in vitro with nanomolar affinity. For ex vivo visualization, Col1-TRAP is labeled with a near-infrared fluorescent dye (NIR-Col1-TRAP). Both Col1-TRAP and NIR-Col1-TRAP display approximately a 3.8-fold greater binding to type I collagen compared to TRAP when measured by surface plasmon resonance (SPR). We present a proof-of-concept study using NIR-Col1-TRAP to detect fibrotic type I collagen deposition ex vivo in the livers of mice with non-alcoholic steatohepatitis (NASH). We show that NIR-Col1-TRAP demonstrates significantly decreased plasma recirculation time as well as increased liver accumulation in the NASH mice compared to mice without disease over 4 hours. As a result, NIR-Col1-TRAP shows potential as an imaging probe for NASH with in vivo targeting performance after injection in mice. Statement of significanceDirect molecular imaging of fibrosis in NASH patients enables the diagnosis and monitoring of disease progression with greater specificity and resolution than do elastography-based methods or blood tests. In addition, protein-based imaging probes are more advantageous than alternatives due to their biodegradability and scalable biosynthesis. With the aid of computational modeling, we have designed a self-assembled protein micelle that binds to fibrillar and monomeric collagen in vitro. After the protein was labeled with near-infrared fluorescent dye, we injected the compound into mice fed on a NASH diet. NIR-Col1-TRAP clears from the serum faster in these mice compared to control mice, and accumulates significantly more in fibrotic livers.This work advances the development of targeted protein probes for in vivo fibrosis imaging.

Topical Nanoliposomal Collagen Delivery for Targeted Fibril Formation by Electrical Stimulation.

Collagen is a complex, large protein molecule that presents a challenge in delivering it to the skin due to its size and intricate structure. However, conventional collagen delivery methods are either invasive or may affect the protein's structural integrity. This study introduces a novel approach involving the encapsulation of collagen monomers within zwitterionic nanoliposomes, termed Lip-Cols, and the controlled formation of collagen fibrils through electric fields (EF) stimulation. The results reveal the self-assembly process of Lip-Cols through electroporation and a pH gradient change uniquely triggered by EF, leading to the alignment and aggregation of Lip-Cols on the electrode interface. Notably, Lip-Cols exhibit the capability to direct the orientation of collagen fibrils within human dermal fibroblasts. In conjunction with EF, Lip-Cols can deliver collagen into the dermal layer and increase the collagen amount in the skin. The findings provide novel insights into the directed formation of collagen fibrils via electrical stimulation and the potential of Lip-Cols as a non-invasive drug delivery system for anti-aging applications.

Elucidating the role of water in collagen self-assembly by isotopically modulating collagen hydration

Water is known to play an important role in collagen self-assembly, but it is still largely unclear how water-collagen interactions influence the assembly process and determine the fibril network properties. Here, we use the H[Formula: see text]O/D[Formula: see text]O isotope effect on the hydrogen-bond strength in water to investigate the role of hydration in collagen self-assembly. We dissolve collagen in H[Formula: see text]O and D[Formula: see text]O and compare the growth kinetics and the structure of the collagen assemblies formed in these water isotopomers. Surprisingly, collagen assembly occurs ten times faster in D[Formula: see text]O than in H[Formula: see text]O, and collagen in D[Formula: see text]O self-assembles into much thinner fibrils, that form a more inhomogeneous and softer network, with a fourfold reduction in elastic modulus when compared to H[Formula: see text]O. Combining spectroscopic measurements with atomistic simulations, we show that collagen in D[Formula: see text]O is less hydrated than in H[Formula: see text]O. This partial dehydration lowers the enthalpic penalty for water removal and reorganization at the collagen-water interface, increasing the self-assembly rate and the number of nucleation centers, leading to thinner fibrils and a softer network. Coarse-grained simulations show that the acceleration in the initial nucleation rate can be reproduced by the enhancement of electrostatic interactions. These results show that water acts as a mediator between collagen monomers, by modulating their interactions so as to optimize the assembly process and, thus, the final network properties. We believe that isotopically modulating the hydration of proteins can be a valuable method to investigate the role of water in protein structural dynamics and protein self-assembly.

Graft copolymers of collagen and acrylic monomers—Reagents for sizing of cotton yarn

AbstractA new highly effective sizing reagent based on grafted collagen copolymers has been proposed for cotton yarn. The copolymers were synthesized by reacting an aqueous solution of collagen with acrylic monomers—acrylic, matacrylic acids, and their amides in the presence of potassium persulfate. Based on Scanning electron microscopy ‐ Energy dispersive (SEM‐EDS) analysis data, the composition of the copolymers was determined. Unsaturated acids are more actively grafted onto collagen than their amides. When a polymer film is formed from a solution of a collagen copolymer with (meth)acrylic acid, microcracks appear on the surface of the sample; microcracks are absent in films of copolymers with (meth)acrylamide. Films of copolymers, especially methacrylamide, are quite flexible and contribute to a significant improvement in the properties of the yarn. The tensile strength of yarn sized with copolymers increases by 20%–65% compared to unsized yarn, and by 13%–38% compared to yarn sized with a starch solution. At the same time, the relative tensile elongation of the experimental yarn improves by 14%–58%, while this property of starch‐size yarn deteriorates by 14%–22% compared to unsized yarn. The complete removal of graft copolymers from the surface of the yarn in the process of biochemical desizing using the enzyme protease is shown.Highlights Graft copolymerization of (meth)acrylamide with collagen was carried out. Grafting is initiated by the interaction of collagen with potassium persulfate. The synthesized copolymers are effective sizing reagents for cotton yarn. The copolymer with poly(meth)acrylamide forms a flexible and stable film. The protease enzyme completely removes the size from the surface of the yarn.

Targeting Intratibial Osteosarcoma Using Water-Soluble Copolymers Conjugated to Collagen Hybridizing Peptides.

Osteosarcoma (OS) is the most common form of primary malignant bone cancer in adolescents. Over the years, OS prognosis has greatly improved due to adjuvant and neoadjuvant (preoperative) chemotherapeutic treatment, increasing the chances of successful surgery and reducing the need for limb amputation. However, chemotherapeutic treatment to treat OS is limited by off-target toxicities and requires improved localization at the tumor site. Collagen, the main constituent of bone tissue, is extensively degraded and remodeled in OS, leading to an increased availability of denatured (monomeric) collagen. Collagen hybridizing peptides (CHPs) comprise a class of peptides rationally designed to selectively bind to denatured collagen. In this work, we have conjugated CHPs as targeting moieties to water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers to target OS tumors. We demonstrated increased accumulation of collagen-targeted HPMA copolymer-CHP conjugates compared to nontargeted HPMA copolymers, as well as increased retention compared to both nontargeted copolymers and CHPs, in a murine intratibial OS tumor model. Furthermore, we used microcomputed tomography analysis to evaluate the bone microarchitecture and correlated bone morphometric parameters (porosity, bone volume, and surface area) with maximum accumulation (Smax) and accumulation at 168 h postinjection (S168) of the copolymers at the tumor. Our results provide the foundation for the use of HPMA copolymer-CHP conjugates as targeted drug delivery systems in OS tumors.

A delivery system into the dermis layer of skin using small unilamellar liposomes and targeted collagen regeneration stimulated by electric field

Collagen is one of the prominent scaffolding components of the extracellular matrix that can turn into collagen fibers through self-assembly mechanisms. Understanding that various factors can influence collagen assembly, we studied the effect of electric field exposure and visualized collagen fibrils' fibrillogenesis process under the confocal microscope. Collagen monomers started to aggregate within 10 seconds after applying an AC of 10 Hz-1V, and collagen fibrils with a 28- to 70-nm diameter appeared within 9 minutes. Further, we designed an experiment to form collagen fibers at a targeted region by encapsulating collagen molecules in nano-sized liposomes (Lip-Col) and have confirmed that the fibrils were successfully formed. We infer that the electric field could trigger the electrical interaction between hydronium ion and the hydroxyl group of hydroxyproline in the collagen peptide chains, resulting in the change of the surface charge of the collagen and pH solution. In vitro, Lip-Cols can interact with the cellular membrane of human dermal fibroblast and form collagen networks in extracellular matrix space. Our findings can potentially be utilized as a drug delivery system for anti-aging or wound healing agents.

Studying on effects of boiling on texture, microstructure and physiochemical properties of sea cucumber body wall and its mechanism using second harmonic generation (SHG) microscopy

The structural foundation of texture changes in sea cucumber body wall (SCBW) during boiling was investigated using second harmonic generation (SHG) microscopy for the first time. The results from SHG signal imaging, light microscopy, scanning electron microscopy and transmission electron microscopy indicated the hierarchical structures of collagen in SCBW underwent progressive destruction with the prolongation of boiling time, including the depolymerization of collagen fibres, the uncoiling and disaggregation of collagen fibrils, the destruction of collagen microfibrils, the loosing of triple helix structure of collagen, and the degradation and gelatinization of collagen, which contributed to the progressive decline in texture indicators including shear force and hardness. SHG analysis also indicated that although collagen macromolecules such as collagen fibres, collagen fibrils and collagen microfibrils could be observed in 0.5 h-boiled and 2 h-boiled SCBW, monomeric collagen, the basic structural components of those macromolecules, has been already damaged.

A Bilayer Osteochondral Scaffold with Self‐Assembled Monomeric Collagen Type‐I, Type‐II, and Polymerized Chondroitin Sulfate Promotes Chondrogenic and Osteogenic Differentiation of Mesenchymal Stem Cells

Osteochondral (OC) injuries are suffered by over 40 million patients in Europe alone. Tissue‐engineering approaches may provide a more promising alternative over current treatments by potentially eliminating the need for revision surgery and creating a long‐term substitute. Herein, the goal is to capture the natural biological and mechanical properties of the joint by developing a bilayer scaffold that is novel in two ways: first, a biomimetic bottom‐up approach is used to improve production precision and reduce immunogenicity; monomeric collagen type I and II are self‐assembled to fibrils and then processed to 3D scaffolds. Second, to induce a tissue‐specific response in mesenchymal stem cells (MSCs), polymerized chondroitin sulfate (PCS) is synthesized and grafted to collagen II and hydroxyapatite (HA) is added to collagen I. Incorporation of PCS into collagen II induces a chondrogenic response by upregulation of COL2A1 and ACAN expression, and incorporation of HA into collagen I stimulates osteogenesis and upregulates the expression of COL1A2 and RUNX2. It is remarkable that MSCs give rise to distinct behavior of chondrogenesis and osteogenesis in the two different regions of the bilayer scaffold. This hybrid scaffold of collagen II‐PCS and collagen I‐HA offers a great potential treatment for OC injuries.

Design and Production of Customizable and Highly Aligned Fibrillar Collagen Scaffolds.

The ability to fabricate anisotropic collagenous materials rapidly and reproducibly has remained elusive despite decades of research. Balancing the natural propensity of monomeric collagen (COL) to spontaneously polymerize in vitro with the mild processing conditions needed to maintain its native substructure upon polymerization introduces challenges that are not easily amenable with off-the-shelf instrumentation. To overcome these challenges, we have designed a platform that simultaneously aligns type I COL fibrils under mild shear flow and builds up the material through layer-by-layer assembly. We explored the mechanisms propagating fibril alignment, targeting experimental variables such as shear rate, viscosity, and time. Coarse-grained molecular dynamics simulations were also employed to help understand how initial reaction conditions including chain length, indicative of initial polymerization, and chain density, indicative of concentration, in the reaction environment impact fibril growth and alignment. When taken together, the mechanistic insights gleaned from these studies inspired the design, iteration, fabrication, and then customization of the fibrous collagenous materials, illustrating a platform material that can be readily adapted to future tissue engineering applications.

Compositional and structural characteristics of pepsin-soluble type I collagen from the scales of red drum fish, Sciaenops ocellatus

The type I pepsin-soluble collagen (type I PSC) from the red drum fish (Sciaenops ocellatus) scales was successfully purified by hydrophilic ultrafiltration (UF). The collagen purification process was appraised in depth and described in detail, respectively. Based on SDS-PAGE analysis, using proteomics analysis and Isoform-sequencing (Iso-Seq), it was confirmed to be the type I PSC, containing collagen α1 and α2 chains as the single components, with the absence of the non- collagenous proteins, non-type I collagens and potentially allergenic proteins. The type I PSC was verified to be non-denatured collagen monomers with integrated triple-helical structure using gel filtration chromatography (GFC), ion-exchange chromatography (IEC), reversed-phase HPLC (RP-HPLC), fourier transform infrared spectroscopy (FTIR), matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) and amino acid composition analysis. Thus, the accurate primary structures of the type I PSC with 100% matched peptide coverage were obtained for the first time. Additionally, our current study is the first to illustrate the protein component changes in collagen induced by hydrophilic UF and the primary structure changes in collagen molecules induced by pepsin solubilization.

Method for measurement of collagen monomer orientation in fluorescence microscopy.

.Significance: Collagen is the most abundant protein in vertebrates and is found in tissues that regularly experience tension, compression, and shear forces. However, the underlying mechanism of collagen fibril formation and remodeling is poorly understood.Aim: We explore how a collagen monomer is visualized using fluorescence microscopy and how its spatial orientation is determined. Defining the orientation of collagen monomers is not a trivial problem, as the monomer has a weak contrast and is relatively small. It is possible to attach fluorescence tags for contrast, but the size is still a problem for detecting orientation using fluorescence microscopy.Approach: We present two methods for detecting a monomer and classifying its orientation. A modified Gabor filter set and an automatic classifier trained by convolutional neural network based on a synthetic dataset were used.Results: By evaluating the performance of these two approaches with synthetic and experimental data, our results show that it is possible to determine the location and orientation with an error of of a single monomer with fluorescence microscopy.Conclusions: These findings can contribute to our understanding of collagen monomers interaction with collagen fibrils surface during fibril formation and remodeling.

State of collagenolysis in experimental periodontitis of bacterial-immune genesis and its correction with flavonol

The article presents an assessment of the dynamics of changes in the content of the marker of collagenolysis – free oxyproline in the homogeniate of soft tissues and bone in experimental bacterial-immune periodontitis and elucidation of the effect of flavonol quercetin on these indicators. The aim of this study was to determine the role of cytokinogenesis and the effect of flavonol on it in the pathogenesis, development and course of experimental periodontitis. During the experiment, a fragment of the mandible was taken from the animals, from which the soft tissues and bone were carefully separated. The state of collagen was determined by the content of free oxyproline in the soft and bone tissues. The concentration was determined according to the calibration graph and expressed in μmol/g. The results of studies of the indicators of the state of biopolymers of connective tissue structures of periodontium on the 7th, 14th and 30th day of experimental bacterial-immune periodontitis and after its correction with flavonol (from the 7th to the 14th day of the experiment) are presented. The data on the nature of changes in the content of collagen monomers in the process of formation of the inflammatory focus in the periodontal complex are given. During the acute phase of the inflammatory process in rats there was revealed a slight increase in blood free oxyproline in bone homogenate and homogenate of soft periodontal tissues, on the 14th day the dynamics continued to increase, at a later stage of the experiment, namely on the 30th day, increase in bone resorption continued as compared to the 7th and 14th day. During the correction of disorders resulted from the development of this pathological process there was a decrease in the level of free oxyproline in the bone homogenate and homogenate of soft tissues of mandibular periodontium, as compared to the same indicators of animals who did not receive quercetin on the 14th day. The use of flavonol quercetin, which, by affecting immune processes, limited the inflammatory response in periodontal tissues and stabilized collagenolysis processes in periodontal tissues was manifested by a decrease in free oxyproline in bone and soft tissue homogenates of experimental animals.

Performance of a Universal Adhesive on Etched and Non-Etched Surfaces: Changes in Dentin Permeability

Hybridization process for dentin is well known, but remains a difficult challenge as it is dependent on many factors related to dentin substrate, adhesive nature including its solvent. The development of universal adhesives followed the all-in-one concept of existing one-step self-etch adhesives, thus requiring water in their formulation to ionize hydrophilic acid monomers. Most often, carboxylate and/or phosphate groups are their primary functional monomers [1]. Jee, et al. published in 2016 that ethanol molecules penetrate via increasing solvent accessible surfaces induced by the rearrangement of collagen monomers in the gap region [2]. The aim of this study is to evaluate the discrepancy in dentin permeability after application of a universal adhesive used in self-etching (SE) mode or etch and rinse (E&R) mode, by means of hydraulic conductance measurement.

Measuring collagen fibril diameter with differential interference contrast microscopy

Collagen fibrils, linear arrangements of collagen monomers, 20–500 nm in diameter, comprising hundreds of molecules in their cross-section, are the fundamental structural unit in a variety of load-bearing tissues such as tendons, ligaments, skin, cornea, and bone. These fibrils often assemble into more complex structures, providing mechanical stability, strength, or toughness to the host tissue. Unfortunately, there is little information available on individual fibril dynamics, mechanics, growth, aggregation and remodeling because they are difficult to image using visible light as a probe. The principle quantity of interest is the fibril diameter, which is difficult to extract accurately, dynamically, in situ and non-destructively. An optical method, differential interference contrast (DIC) microscopy has been used to visualize dynamic structures that are as small as microtubules (25 nm diameter) and has been shown to be sensitive to the size of objects smaller than the wavelength of light. In this investigation, we take advantage of DIC microscopy’s ability to report dimensions of nanometer scale objects to generate a curve that relates collagen diameter to DIC edge intensity shift (DIC-EIS). We further calibrate the curve using electron microscopy and demonstrate a linear correlation between fibril diameter and the DIC-EIS. Using a non-oil immersion, 40x objective (NA 0.6), collagen fibril diameters between ~100 nm to ~ 300 nm could be obtained with ±11 and ±4 nm accuracy for dehydrated and hydrated fibrils, respectively. This simple, nondestructive, label free method should advance our ability to directly examine fibril dynamics under experimental conditions that are physiologically relevant.

Interaction between KDELR2 and HSP47 as a Key Determinant in Osteogenesis Imperfecta Caused by Bi-allelic Variants in KDELR2

Osteogenesis imperfecta (OI) is characterized primarily by susceptibility to fractures with or without bone deformation. OI is genetically heterogeneous: over 20 genetic causes are recognized. We identified bi-allelic pathogenic KDELR2 variants as a cause of OI in four families. KDELR2 encodes KDEL endoplasmic reticulum protein retention receptor 2, which recycles ER-resident proteins with a KDEL-like peptide from the cis-Golgi to the ER through COPI retrograde transport. Analysis of patient primary fibroblasts showed intracellular decrease of HSP47 and FKBP65 along with reduced procollagen type I in culture media. Electron microscopy identified an abnormal quality of secreted collagen fibrils with increased amount of HSP47 bound to monomeric and multimeric collagen molecules. Mapping the identified KDELR2 variants onto the crystal structure of G.gallus KDELR2 indicated that these lead to an inactive receptor resulting in impaired KDELR2-mediated Golgi-ER transport. Therefore, in KDELR2-deficient individuals, OI most likely occurs because of the inability of HSP47 to bind KDELR2 and dissociate from collagen type I. Instead, HSP47 remains bound to collagen molecules extracellularly, disrupting fiber formation. This highlights the importance of intracellular recycling of ER-resident molecular chaperones for collagen type I and bone metabolism and a crucial role of HSP47 in the KDELR2-associated pathogenic mechanism leading to OI.

A Bottom-Up Approach Grafts Collagen Fibrils Perpendicularly to Titanium Surfaces.

Currently, titanium dental implant apposition to bone is achieved via osseointegration leading to ankylosis. A biomimetic Sharpey's fiber-type interface could be constructed around collagen fibrils robustly attached and projecting perpendicularly from the titanium surface. We present a proof-of-concept for a method to create upright-standing collagen nanofibrils covalently bonded to a titanium surface. The method involves activation of the titanium surface using a plasma discharge treatment followed by functionalization with an oxyamine-terminated silane coupling molecule. Using Rapoport's salt, the N-termini of individual type I collagen monomers are converted to ketones. When presented to the functionalized titanium surface, these ketones form oxime linkages with the silanes thus immobilizing the collagen. In a two-step process, these covalently bonded monomers act as sites for the formation of fibrils. Many fibril-surface junctions were observed by scanning electron microscopy on three different surfaces. These findings set the stage for working toward a high surface density of such features which might act as a platform from which to build a synthetic ligament.

A fundamental insight on developing biological value product from plant wastes

Collagen has significant contributions to the field of biomedical and tissue engineering applications. Native collagen biomaterials are mechanically feeble and require stabilization against enzymes for multivariate applications. In this study, phytoconstituents are extracted from Artemisia pallens and interacted with collagen for enhancing its chemical properties for biomedical use. From FTIR analysis, it is evident that Artemisia pallens influences the conformational changes, and this change does not affect the stability of collagen, which is ascertained by the electrophoretic study. In the fibrillogenesis study, there is a decrease in the T½ time, which indicates the reduction in the gelling time due to the influence of Artemisia pallens. Histology characterization also confirms the formation of fibrils under the control of Artemisia pallens at a pH < pI. Moreover, anti-glycation activity has been enhanced at a higher concentration owing to its antioxidant characteristics of the phytoconstituents. From the results, it is inferred that influence of Artemisia pallens creates a nucleation center for the monomeric collagen to aggregate in an uniform order, thus stimulating fibrillogenesis and along with its antiglycation activity, Artemisia pallens can assist in establishing a new way in collagen-based biomaterial preparation.

The Effect of Cholesterol on Membrane Binding and Self-Assembly of Collagen Fibrils.

Collagen is a skeleton of native extracellular matrix (ECM) that is known to provide mechanical and structural stability. In an attempt to develop a new connective cellular model with the surrounding ECM without further experimental complications, such as the reconstitution of ECM receptors, we designed the experiments and discovered that the fibrillogenesis of membrane-bound collagen is not spontaneous as it is in the form of free collagen in bulk solution. The confocal microscopic results suggest that cholesterol is a crucial component that facilitates the fibril formation on the membrane surface. In situ X-ray and neutron reflectivity on Langmuir monolayer and solid-supported lipid bilayer models, respectively, reveal two features of cholesterol effects on the collagen fibril formation. Mainly, cholesterol increases the lateral lipid headgroup separation on the membrane surface, which promotes the association degree of collagen monomers. It also enhances the elastic modulus of the membrane to impede membrane filtration by the collagen assemblies.